Re: [ccp4bb] Stuck rfree - possible non merohedral twinning ?
Hi Juergen other experts Thanks for the suggestions. I was under the impression that the twin laws/operators are to be used if the twinning is merohedral. In my case, it appears as if the twinning is non-merohedral and more over the data i have is processed as P422 which does not have twin operators ( probably because all the axial pairs are same anyway). Probably because of the same reason, xtriage did not give any operators for me to use. In such cases, do people usually try to process the data in another space group of lower symmetry which has a twin law and is there a definite way to distinguish twinning by merohedry vs non-merohedry other than just looking at the diffraction pattern ( is it crucial to make that differentiation ?) ? Thanks Mahesh On Sun, Aug 25, 2013 at 10:24 PM, Bosch, Juergen jubo...@jhsph.edu wrote: Hi Mahesh, if you use Refmac, then you can tell it to refine the twin fraction, no need to tell it the twin law as Refmac will figure it out. If you use phenix, you explicitly tell it the twin law and refine then with it. You can get the possible twin laws by running phenix.xtriage and looking at the log file. For a 1.7 Å dataset you should see excellent holes in the Phe and Tyr, even though your Rfactors are high. If that is the case then you are likely correct with the twin (if nothing else is wrong, Cbeta, Ramas etc). And you did add some waters to your structure already right ? if not the go water picking via Coot. Have you been converted to XDS now ? Welcome to the club. Jürgen On Aug 25, 2013, at 8:35 PM, Mahesh Lingaraju wrote: Hello everyone, I collected a dataset which looked like it is twinned ( or a really long axis in the cell) and did not process in HKL2000 and MOSFLM but with some of help and suggestions from CCP4BB, XDS was able to process it. The data looks good upto 1.7 Å. However, the rfree is stuck at 0.34 even though my model is almost complete. I am beginning to wonder if the data is really twinned as it has the characteristics of non- merohedral twinning: In the images some of the reflections are sharp while some are split and one of the axis in the cell is usually long ( the cell is a= 46.78 b= 46.78 c= 400.34; 90 90 90) is there anyway to work around this ? or collecting better data is the only solution ? Any help is deeply appreciated Thanks Mahesh .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
Re: [ccp4bb] Stuck rfree - possible non merohedral twinning ?
Hi Mahesh You might find this paper useful for some background on types of twinning. It explains the difference between merohedral and non-merohedral twinning. http://journals.iucr.org/d/issues/2003/11/00/ba5036/index.html Alice -- Alice Dawson WNH Lab Division of Biological Chemistry and Drug Discovery College of Life Sciences University of Dundee 01382 385744 From: Mahesh Lingaraju mxl1...@psu.edumailto:mxl1...@psu.edu Reply-To: Mahesh Lingaraju mxl1...@psu.edumailto:mxl1...@psu.edu Date: Monday, 26 August 2013 16:43 To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Stuck rfree - possible non merohedral twinning ? Hi Juergen other experts Thanks for the suggestions. I was under the impression that the twin laws/operators are to be used if the twinning is merohedral. In my case, it appears as if the twinning is non-merohedral and more over the data i have is processed as P422 which does not have twin operators ( probably because all the axial pairs are same anyway). Probably because of the same reason, xtriage did not give any operators for me to use. In such cases, do people usually try to process the data in another space group of lower symmetry which has a twin law and is there a definite way to distinguish twinning by merohedry vs non-merohedry other than just looking at the diffraction pattern ( is it crucial to make that differentiation ?) ? Thanks Mahesh On Sun, Aug 25, 2013 at 10:24 PM, Bosch, Juergen jubo...@jhsph.edumailto:jubo...@jhsph.edu wrote: Hi Mahesh, if you use Refmac, then you can tell it to refine the twin fraction, no need to tell it the twin law as Refmac will figure it out. If you use phenix, you explicitly tell it the twin law and refine then with it. You can get the possible twin laws by running phenix.xtriage and looking at the log file. For a 1.7 Å dataset you should see excellent holes in the Phe and Tyr, even though your Rfactors are high. If that is the case then you are likely correct with the twin (if nothing else is wrong, Cbeta, Ramas etc). And you did add some waters to your structure already right ? if not the go water picking via Coot. Have you been converted to XDS now ? Welcome to the club. Jürgen On Aug 25, 2013, at 8:35 PM, Mahesh Lingaraju wrote: Hello everyone, I collected a dataset which looked like it is twinned ( or a really long axis in the cell) and did not process in HKL2000 and MOSFLM but with some of help and suggestions from CCP4BB, XDS was able to process it. The data looks good upto 1.7 Å. However, the rfree is stuck at 0.34 even though my model is almost complete. I am beginning to wonder if the data is really twinned as it has the characteristics of non- merohedral twinning: In the images some of the reflections are sharp while some are split and one of the axis in the cell is usually long ( the cell is a= 46.78 b= 46.78 c= 400.34; 90 90 90) is there anyway to work around this ? or collecting better data is the only solution ? Any help is deeply appreciated Thanks Mahesh .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742tel:%2B1-410-614-4742 Lab: +1-410-614-4894tel:%2B1-410-614-4894 Fax: +1-410-955-2926tel:%2B1-410-955-2926 http://lupo.jhsph.edu The University of Dundee is a registered Scottish Charity, No: SC015096
Re: [ccp4bb] Stuck rfree - possible non merohedral twinning ?
Dear Mahesh, from the images you showed a few days ago I am not convinced the issue is twinning, just overlaps due to the long c-axis. But of course, I do not have have as much info as you, just those images. Unless you are really convinced you have twinning, if you can see what you want to see in the density, perhaps just do not worry too much about it. Or try to collect some better data, with no overlaps, from a crystal mounted with the long axis along the rotation axis, others and us have collected some good data on these kinds of crystals this way. XDS may have integrated your current images reasonably well, but if the crystal is mounted better (mini-kappa goniometer for instance, or a bent loop, or mount many crystals and trust on luck), it will be able to do an even better job. Mark On 26 Aug 2013, at 17:43, Mahesh Lingaraju wrote: Hi Juergen other experts Thanks for the suggestions. I was under the impression that the twin laws/operators are to be used if the twinning is merohedral. In my case, it appears as if the twinning is non-merohedral and more over the data i have is processed as P422 which does not have twin operators ( probably because all the axial pairs are same anyway). Probably because of the same reason, xtriage did not give any operators for me to use. In such cases, do people usually try to process the data in another space group of lower symmetry which has a twin law and is there a definite way to distinguish twinning by merohedry vs non-merohedry other than just looking at the diffraction pattern ( is it crucial to make that differentiation ?) ? Thanks Mahesh On Sun, Aug 25, 2013 at 10:24 PM, Bosch, Juergen jubo...@jhsph.edu wrote: Hi Mahesh, if you use Refmac, then you can tell it to refine the twin fraction, no need to tell it the twin law as Refmac will figure it out. If you use phenix, you explicitly tell it the twin law and refine then with it. You can get the possible twin laws by running phenix.xtriage and looking at the log file. For a 1.7 Å dataset you should see excellent holes in the Phe and Tyr, even though your Rfactors are high. If that is the case then you are likely correct with the twin (if nothing else is wrong, Cbeta, Ramas etc). And you did add some waters to your structure already right ? if not the go water picking via Coot. Have you been converted to XDS now ? Welcome to the club. Jürgen On Aug 25, 2013, at 8:35 PM, Mahesh Lingaraju wrote: Hello everyone, I collected a dataset which looked like it is twinned ( or a really long axis in the cell) and did not process in HKL2000 and MOSFLM but with some of help and suggestions from CCP4BB, XDS was able to process it. The data looks good upto 1.7 Å. However, the rfree is stuck at 0.34 even though my model is almost complete. I am beginning to wonder if the data is really twinned as it has the characteristics of non- merohedral twinning: In the images some of the reflections are sharp while some are split and one of the axis in the cell is usually long ( the cell is a= 46.78 b= 46.78 c= 400.34; 90 90 90) is there anyway to work around this ? or collecting better data is the only solution ? Any help is deeply appreciated Thanks Mahesh .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
Re: [ccp4bb] Stuck rfree - possible non merohedral twinning ?
Hi Mahesh, First of all, I risk going out on a limb here since I have no demonstrable experience concerning your topic. I was trying to solve a non-merohedral dataset for years, and I failed (like failure as defined in the dictionary). That is hardly a reference, but I got to read a little on the topic and maybe I got some comments that can be useful for you. So, if you data would be twinned by non-merohedry, you would not have a valid solution by now. You would have only a fraction of the reflections indexed and a significant fraction in that index would be overlaps. The others would be partial overlaps/splits and the rest fully split reflections (singlets). Solving a non-merohedrally twinned structure requires developing an independent crystal rotation matrix for each twin domain, integrating in the correct point group, leaving all related reflections unmerged and with the original index, solving the structure and identifying the twin law (the one that describes the superposition of one twin domain on top of the other one) during structure solution/refinement. According to my knowledge, this has not been done with a novel structure as of now. (As for anything I say here, someone please educate me if I am wrong) The really mean part with non-merohedral data is, that you cannot even index the data properly, although a variety of indexing choices will give you reasonably well integration/merging statistics and you might not notice it in the beginning. It is very hard to identify the correct cell. A rough guideline is, that in many cases, crystals twinned by non-merohedry display a relationship of a~2b (2a~b, depending on choice) so you can try to double the axis for either a or b in a tetragonal cell and see if your predictions for a primitive cell with those dimensions overlap better with your reflections. The true space group of the lattice will emerge at a later point of time. The best way to do the indexing is supposedly using programs for small-molecule crystallography. When (=if) you have the two matrices, you can forward them to your protein x-ray crystallography software and integrate twice using one matrix at a time. A much better way to deal with non-merohedry is to make different crystals. Or to optimize the cryo. If your data is twinned by pseudo-merohedry, most of the reflections will be overlaps with a very few splits, rather in higher resolution bins than in lower ones. In case your data is really twined, I suspect that is what you are dealing with here by looking at the diffraction patterns you showed before. This means you can solve your structure by integrating your data with only one crystal rotation matrix. However, your data might appear to be not only in a higher point group but also in a higher lattice system than it actually is. Your data then emulates a P422 but your structure does not generate the crystal by that symmetry relationship. Since there is no twin law for P422, phenix.xtriage can not help you there with merged data. Treating twinning by pseudo-merohedry is pretty much straight forward. You must process your data in the correct point group and later identify and apply the twin law. Start with P1 and work up if you have enough frames. Also, if you have more than one molecule in the ASU, you might have simply overestimated the symmetry. I have seen this once in a monoclinic cell with two copies in the ASU where one angle was ~90.08, but not 90.00. It still merged reasonably well as an orthorombic cell, but it was not twined. In either case, try processing your images in a lower point group first, and look carefully at your data. Did your predicted reflections cover all the observed reflections? Did you predict reflections (entire lunes?) that are not there? How does your native Patterson map look like if you process your data in P1? good luck, s.
[ccp4bb] Stuck rfree - possible non merohedral twinning ?
Hello everyone, I collected a dataset which looked like it is twinned ( or a really long axis in the cell) and did not process in HKL2000 and MOSFLM but with some of help and suggestions from CCP4BB, XDS was able to process it. The data looks good upto 1.7 Å. However, the rfree is stuck at 0.34 even though my model is almost complete. I am beginning to wonder if the data is really twinned as it has the characteristics of non- merohedral twinning: In the images some of the reflections are sharp while some are split and one of the axis in the cell is usually long ( the cell is a= 46.78 b= 46.78 c= 400.34; 90 90 90) is there anyway to work around this ? or collecting better data is the only solution ? Any help is deeply appreciated Thanks Mahesh
Re: [ccp4bb] Stuck rfree - possible non merohedral twinning ?
Hi Mahesh, if you use Refmac, then you can tell it to refine the twin fraction, no need to tell it the twin law as Refmac will figure it out. If you use phenix, you explicitly tell it the twin law and refine then with it. You can get the possible twin laws by running phenix.xtriage and looking at the log file. For a 1.7 Å dataset you should see excellent holes in the Phe and Tyr, even though your Rfactors are high. If that is the case then you are likely correct with the twin (if nothing else is wrong, Cbeta, Ramas etc). And you did add some waters to your structure already right ? if not the go water picking via Coot. Have you been converted to XDS now ? Welcome to the club. Jürgen On Aug 25, 2013, at 8:35 PM, Mahesh Lingaraju wrote: Hello everyone, I collected a dataset which looked like it is twinned ( or a really long axis in the cell) and did not process in HKL2000 and MOSFLM but with some of help and suggestions from CCP4BB, XDS was able to process it. The data looks good upto 1.7 Å. However, the rfree is stuck at 0.34 even though my model is almost complete. I am beginning to wonder if the data is really twinned as it has the characteristics of non- merohedral twinning: In the images some of the reflections are sharp while some are split and one of the axis in the cell is usually long ( the cell is a= 46.78 b= 46.78 c= 400.34; 90 90 90) is there anyway to work around this ? or collecting better data is the only solution ? Any help is deeply appreciated Thanks Mahesh .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
